Novel strain of streptomyces for controlling plant diseases

ABSTRACT

A novel antibiotic-producing Streptomyces sp. is provided that exhibits antifungal activity only on certain specific plant pathogens. Also provided is a method of treating or protecting plants from fungal infections comprising applying an effective amount of an antibiotic-producing Streptomyces sp. having all the identifying characteristics of NRRL Accession number B-30145. The invention also relates to fungicidal compositions comprising this novel Streptomyces strain and the antibiotics and metabolites produced by this strain either alone, or in combination with other chemical and biological pesticides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/671,943 filed Sep. 27, 2000. The contents of thisapplication is hereby incorporated by reference into the presentdisclosure.

FIELD OF THE INVENTION

[0002] The present invention is in the field of biopesticides.

BACKGROUND OF THE INVENTION

[0003] For a number of years, it has been known that variousmicroorganisms exhibit biological activity so as to be useful to controlplant diseases. Although progress has been made in the field ofidentifying and developing biological pesticides for controlling variousplant diseases of agronomic and horticultural importance, most of thepesticides in use are still synthetic compounds. Many of these chemicalfungicides are classified as carcinogens by the EPA and are toxic towildlife and other non-target species. In addition, pathogens maydevelop resistance to chemical pesticides (Schwinn et al., 1991).

[0004] Biological control offers an attractive alternative to syntheticchemical fungicides. Biopesticides (living organisms and the compoundsnaturally produced by these organisms) can be safer, more biodegradable,and less expensive to develop.

[0005] The actinomycetes, including the streptomycetes, are knownproducers of antifungal metabolites (Lechavalier and Waksman, 1962;Lechavalier, 1988). Several 25 actinomycete-produced antibiotics areroutinely used in an agricultural setting such as streptomycin andterramycin for fire blight control.

[0006] Streptomycetes have demonstrated both in vitro and in vivoactivity against plant pathogens. Axelrood et al. (1996) isolated 298actinomycetes from Douglas-fir roots. Approximately 30% of these strainsdemonstrated antifungal activity against Fusarium, Cylindrocarpon,and/or Pythium in vitro. Yuan and Crawford (1995) reported thatStreptomyces lydicus WYEC 108 showed both strong in vitro antifungalactivity and inhibition of Pythium root rot in pot tests with pea orcotton seed. Reddi and Rao (1971) controlled Pythium damping-off intomatoes and Fusarium wilt of cotton with Streptomyces ambofaciens.Rhizoctonia root rot was controlled by Streptomyces hygroscopicus var.geldanus (Rothrock and Gofflieb, 1984). These authors reported that thecontrol was dependent on the in situ geldanamycin concentration producedby this strain. The same authors also saw protection of soybeans fromPhytophthora megasperma var. sojae by Streptomyces herbaricolor andStreptomyces coeruleofuscus (1984). Chamberlain and Crawford (1999) sawin vitro and in vivo antagonism of turfgrass fungal pathogens by S.hygroscopicus strain YCED9. Crawford (1996) patented the use of thisstrain to control plant pathogens in U.S. Pat. No. 5,527,526. Suh (1998)patented 2 Streptomyces sp. that were active against Rhizoctonia solaniand Phytophthora capsici. A Streptomyces griseoviridis product againstFusarium spp. and other soil pathogens is on the market as Mycostop™.

SUMMARY OF THE INVENTION

[0007] A novel antibiotic-producing Streptomyces sp. is provided thatexhibits antifungal activity only on certain specific plant pathogens.Also provided is a method of treating or protecting plants from fungalinfections comprising applying an effective amount of anantibiotic-producing Streptomyces sp. having all the identifyingcharacteristics of NRRL Accession number B-30145. The invention alsorelates to fungicidal compositions comprising this novel Streptomycesstrain and the antibiotics and metabolites produced by this straineither alone, or in combination with other chemical and biologicalpesticides.

[0008] The antibiotic-producing Streptomyces sp. can be provided as asuspension in a whole broth culture or as an antibiotic-containingsupernatant obtained from a whole broth culture of anantibiotic-producing Streptomyces sp. Also provided is a novelbutanol-soluble antibiotic that exhibits specific antifungal activityand a process for isolating the novel butanol-soluble antibiotic.

BRIEF DESCRIPTION OF THE FIGURES

[0009]FIG. 1A is the analytical HPLC chromatogram of active fraction 6.(Microsorb C18, 10 cm×4.6 mm, 100 Å, flow rate 1 mL/min, UV detection at220 nm, acetonitrile+0.05% TFA/water+0.05% gradient as follows: 0-30min, 5-65%; 30-40 min, 65-100%; 40-45 min, 100%).

[0010]FIG. 1B is the UV spectrum of the active peak eluting at 14.755minutes in the chromatogram described in 1A.

[0011]FIG. 2A is the analytical HPLC chromatogram of active fraction 7under the same conditions described in 1A.

[0012]FIG. 2B is the UV spectrum of the active peak eluting at 16.146minutes in the chromatogram described in 2A.

[0013]FIG. 3 is the C-8 HPLC chromatogram of the methanol eluate fromthe Diaion HP-20 resin step described in Method B. (HP Zorbax EclipsXDB-C8 column, 5 μm, 150×4.6 mm, flow rate 0.8 mL min, UV detection at220 nm, chart speed 2 mm/min. Solvent A, 25:5:70acetonitrile/methanol/water. Solvent B, 65:5:30acetonitrile/methanol/water. Gradient: 100% A at 0 minutes increased to3% B over 20 minutes.)

[0014]FIG. 4 is the ¹H NMR spectrum (400 MHz, CD₃OD) of the semi-pureactive metabolite obtained from purification method A.

[0015]FIG. 5 is the ¹³C NMR spectrum (100 MHz, CD₃OD) of the semi-pureactive metabolite obtained from purification method A.

[0016]FIG. 6 is the LC ESI-MS (Liquid Chromatography ElectroSprayImpact-Mass Spectrum) of Peak A obtained from purification method B.(Microsorb C 18, 10 cm×4.6 mm, 100 Å, flow rate 1 mL/min,acetonitrile+0.02% TFA/water+0.02% gradient as follows: 0-30 min, 5-65%;30-40 min, 65-100%; 40-45 min, 100%).

[0017]FIG. 7 is the LC ESI-MS (Liquid Chromatography ElectroSprayImpact-Mass Spectrum) of Peak B obtained from purification method B.(Microsorb C18, 10 cm×4.6 mm, 100 Å, flow rate 1 mL/min,acetonitrile+0.02% TFA/water+0.02% gradient as follows: 0-30 min, 5-65%;30-40 min, 65-100%; 40-45 min, 100%).

DETAILED DESCRIPTION

[0018] The present invention provides a novel strain of Streptomyces sp.or mutants thereof with antifungal activity only on specific plantpathogens such as Alternaria, Phytophthora, Botrytis, Rhizoctonia andSclerotinia. This novel strain was deposited with the NRRL on Jul. 20,1999 under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure under Accession No. B-30145. The invention also includesmethods of preventing and treating fungal diseases in plants using suchbacterial strains or antibiotic-containing supernatants or pureantibiotics obtained from such bacterial strains. The invention alsoincludes a butanol soluble antifungal antibiotic with a molecular weightof less than 10,000 daltons, with stability to base and to heattreatment of 1 hour at 80° C. and lability to acid treatment.

[0019] Definitions

[0020] The singular form “a,” “an” and “the” include plural referencesunless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

[0021] The term “comprising” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andagriculturally acceptable carriers. “Consisting of” shall mean excludingmore than trace elements of other ingredients and substantial methodsteps for applying the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

[0022] As used herein, “biological control” is defined as control of apathogen or insect by the use of a second organism. Known mechanisms ofbiological control include enteric bacteria that control root rot byout-competing fungi for space on the surface of the root. Bacterialtoxins, such as antibiotics, have been used to control pathogens. Thetoxin can be isolated and applied directly to the plant or the bacterialspecies may be administered so it produces the toxin in situ.

[0023] The term “fungus” or “fungi” includes a wide variety of nucleatedspore-bearing organisms that are devoid of chlorophyll. Examples offungi include yeasts, molds, mildews, rusts, and mushrooms.

[0024] The term “bacteria” includes any prokaryotic organism that doesnot have a distinct nucleus. “Pesticidal” means the ability of asubstance to increase mortality or inhibit the growth rate of plantpests.

[0025] “Fungicidal” means the ability of a substance to increasemortality or inhibit the growth rate of fungi.

[0026] “Antibiotic” includes any substance that is able to kill orinhibit a microorganism. Antibiotics may be produced by a microorganismor by a synthetic process or semisynthetic process. The term, therefore,includes a substance that inhibits or kills fungi for example,cycloheximide or nystatin.

[0027] “Antifungal” includes any substance that is able to kill orinhibit the growth of fungi.

[0028] The term “culturing” refers to the propagation of organisms on orin media of various kinds. “Whole broth culture” refers to a liquidculture containing both cells and media. “Supernatant” refers to theliquid broth remaining when cells grown in broth are removed bycentrifugation, filtration, sedimentation, or other means well known inthe art.

[0029] An “effective amount” is an amount sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations. In terms of treatment and protection, an“effective amount” is that amount sufficient to ameliorate, stabilize,reverse, slow or delay progression of the fungal or bacterial diseasestates. “Positive control” means a compound known to have pesticidalactivity. “Positive controls” include, but are not limited tocommercially available chemical pesticides. The term “negative control”means a compound known not to have pesticidal activity. Examples ofnegative controls are water or ethyl acetate.

[0030] The term “solvent” includes any liquid that holds anothersubstance in solution. “Solvent extractable” refers to any compound thatdissolves in a solvent and which then may be isolated from the solvent.Examples of solvents include, but are not limited to, organic solventslike ethyl acetate.

[0031] The term “metabolite” refers to any compound, substance orbyproduct of a fermentation of a microorganism that has pesticidalactivity. Antibiotic as defined above is a metabolite specificallyactive against a microorganism.

[0032] The term “mutant” refers to a variant of the parental strain aswell as methods for obtaining a mutant or variant in which thepesticidal activity is greater than that expressed by the parentalstrain. The “parent strain” is defined herein as the originalStreptomyces strain before mutagenesis. To obtain such mutants theparental strain may be treated with a chemical such asN-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfone, or byirradiation using gamma, x-ray, or UV-irradiation, or by other meanswell known to those practiced in the art.

[0033] A “composition” is intended to mean a combination of active agentand another compound, carrier or composition, inert (for example, adetectable agent or label or liquid carrier) or active, such as anadjuvant. Examples of agricultural carriers are provided below.

[0034] We describe a novel antibiotic-producing strain of Streptomycessp. NRRL No. B-30145 and mutants thereof that have antifungal activityonly on specific plant pathogens. Also provided is a supernatantisolated from the culture as well as a composition comprising theculture. In a further aspect, the compositions further comprise at leastone chemical or biological pesticide.

[0035] A metabolite produced by the Streptomyces sp. strain is alsoprovided by this invention. The metabolite exhibits activity againstplant pathogenic fungi and is heat and base stable, is acid labile andhas a molecular weight of less than 10,000 daltons. By way of example,the metabolite may have a molecular weight [M+H⁺] between about 925 tobetween about 865.

[0036] The one or more metabolites produced by the Streptomyces sp.strain exhibit UV absorption between about 215 nm and 220 nm. Themetabolite may be comprised of a variety of molecules including, but notlimited to, propargyl alcohol segments [C═C—CH(OH)], oxygenated methinecarbons (X—CH—Y) or a sugar moiety. By way of example, the metabolitemay comprise at least two propargyl segments, several oxygenated methinecarbons (by way of example, e.g., 5 to 10) and/or a sugar moiety.Alternatively, the one or more metabolites produced by the Streptomycessp. strain may share the same carbon skeleton and differ in degree ofoxygenation.

[0037] The present invention also provides antifungal compositionscomprising a metabolite produced by Streptomyces and isolated accordingto a method comprising:

[0038] (a) loading a whole broth culture of Streptomyces sp. strain NRRLNo. B-30145 or mutants thereof having all the identifyingcharacteristics of NRRL No. B-30145 onto a non-ionic absorbent polymericresin;

[0039] (b) eluting the metabolite with an alcohol;

[0040] (c) screening the eluate of step (b) with a bioassay forfractions of the eluate exhibiting antifungal activity;

[0041] (d) loading the fractions of the eluate exhibiting antifungalactivity of step (c) on a HPLC column; and

[0042] (e) eluting the metabolite with an organic solvent.

[0043] The method may further comprise washing the resin with waterprior to step (b) and screening the eluate of step (e) with a bioassayto select the fractions exhibiting antifungal activity.

[0044] The whole broth culture of step (a) may be freeze dried andre-suspended with an aqueous solution (e.g., water) prior to adding tothe non-ionic absorbent polymeric resin. In a preferred embodiment thewhole broth culture added to the resin is a homogenized cell-free wholebroth culture. Examples of non-ionic absorbent polymeric resin that maybe used include, but are not limited to, Supelco Sepabead SP-207 orSupelco Diaon HP-20.

[0045] The eluent used to remove the metabolite in step (b) may be analcohol or a gradient of aqueous alcohol. By way of example, methanol ora gradient of aqueous methanol may be used as the eluent (e.g., Example6).

[0046] The bioassay of step (c) may be any assay which evaluatesantifungal activity. Examples of such bioassays include but are notlimited to, the agar diffusion assay or slide germination assay. Forexample, the bioassay may be a germination assay with Moniliniafructicola and/or Alternaria brassicicola.

[0047] Examples of an HPLC column that may be used in step (d) include,but are not limited to, C-18 or C-8. Examples of the organic solventthat may be used to remove the metabolite from the HPLC column include,but are not limited to, an acetonitrile-water—gradient (e.g., Example6).

[0048] The metabolite can also be formulated as a composition, with acarrier or alternatively, with at least one chemical or biologicalpesticide.

[0049] In order to achieve good dispersion and adhesion of compositionswithin the present invention, it may be advantageous to formulate thewhole broth culture, supernatant and/or metabolite/antibiotic withcomponents that aid dispersion and adhesion. Suitable formulations willbe known to those skilled in the art (wettable powders, granules and thelike, or can be microencapsulated in a suitable medium and the like,liquids such as aqueous flowables and aqueous suspensions, andemulsifiable concentrates). Other suitable formulations will be known tothose skilled in the art.

[0050] The strain, culture, supernatant and isolated metabolite areuseful to protect or treat plants, fruit, and roots from fungalinfections by applying an effective amount of the active formulation tothe plant, fruit or root. The formulations are particularly suited totreat or prevent infections caused by a fungus selected from the groupconsisting of Alternaria solani, Botrytis cinerea, Rhizoctonia sp.,Sclerotinia sp., and Phytophthora sp.

[0051] All patents and publications cited herein are incorporated byreference. Full bibliographic citations for these may be found at theend of the specification, immediately preceding the claims.

[0052] The following examples are provided to illustrate the invention.These examples are not to be construed as limiting.

EXAMPLES Example 1

[0053] Characterization of Strain NRRL No. B-30145

[0054] NRRL No. B-30145 was identified based on 16S rRNA sequencing. Theprotocol used to generate the 16S rRNA gene data sequence (AcculabCustomer Handbook v. 1.0) is described as follows.

[0055] The 16S rRNA gene is PCR amplified from genomic DNA isolated frombacterial colonies. Primers used for the amplification correspond to E.coli positions 005 and 531. Amplification products are purified fromexcess primers and dNTPs using Microcon 100 (Amicon) molecular weightcut-off membranes and checked for quality and quantity by running aportion of the products on an agarose gel.

[0056] Cycle sequencing of the 16S rRNA amplification products iscarried out using AmpliTaq FS DNA polymerase and dRhodamine dyeterminators. Excess dye-labeled terminators were removed from thesequencing reactions using a Sephadex G-50 spin column. The products arecollected by centrifugation, dried under vacuum and frozen at −20° C.until ready to load. Samples are re-suspended in a solution offormamide/blue dextran/EDTA and denatured prior to loading. The samplesare electrophoresed on an ABI Prism 377 DNA Sequencer. Data are analyzedusing PE/Applied Biosystem's DNA editing and assembly software. Onceobtained, sequences are compared against PE/Applied Biosystem'sMicroSeq™ database using MicroSeq™ sequence analysis software. Sequencesare also compared to the GenBank and Ribosomal Database Project (RDP).

[0057] The result of the 16S rRNA sequencing identified NRRL No. B-30145as a Streptomyces sp. This strain may belong to the species S.mashuensis (formerly Streptoverticillium mashuense) or a relatedspecies, as suggested by the sequencing results. The best match wasStreptomyces mashuensis with a 98% match score.

Example 2

[0058] Activity of NRRL No. B-30145 Against Plant Pathogens in In-VitroCulture (Zone Assay).

[0059] NRRL No. B-30145 was tested against an array of different plantpathogens utilizing two different in-vitro assays. The agar diffusion(zone) assay consists of applying either plant pathogen spores over thesurface of an agar medium to create an entire lawn of growth orutilizing a mycelial agar plug placed in the center of the petri dishthat will grow and colonize the agar. Circular wells approximately 7.0mm in diameter are removed from the agar using a pipette attached to avacuum pump. Fermentation samples of NRRL No. B-30145 are added to eachwell along with known standards and water checks. Plates are incubatedfor three to four days under environmental conditions conducive for eachpathogen. Results consist of a zone of no pathogen growth around thewell or a greatly reduced amount of pathogen growth around the well orno affect. The size and type of zone is recorded for each sample.Results for NRRL No. B-30145 in agar diffusion assays are presented inTable 1. Results within agar diffusion were variable; diffusion throughagar may be inhibited. TABLE 1 Activity of NRRL No. B-30145 againstselected plant pathogens in the agar diffusion (zone) assay. Alternariabrassicicola No Zone/Weak Activity Botrytis cinerea Weak ActivityMonilinia fructicola No Zone Phytophthora capsici Moderate activityPythium sp. Weak Activity Colletotrichum acutatum No Zone Rhizoctoniasolani No Zone Sclerotinia sclerotiorum No Zone

[0060] The second type of in-vitro assay performed to test the pathogenspectrum of NRRL No. B-30145 was the slide germination assay.Fermentation samples of NRRL No. B-30145 at various dilutions were addedto glass depression slides (25 mm×75 mm with 18 mm diameter depression1.75 mm deep) and an equal volume of pathogen spores were mixed with thesample. Slides were incubated on moistened paper towels in sealedplastic boxes at room temperature overnight. Results are determined byobserving the fermentation sample/spore suspension sample using acompound microscope at 100×. Typical results consist of lack ofgermination of the pathogen propagule or greatly reduced germinationand/or growth. In addition, various types of malformations of theinitial growth from the pathogen spores may occur. The spectrum ofactivity of NRRL No. B-30145 is presented in Table 2. Completeinhibition of spore germination occurred at low concentrations offermentation samples. TABLE 2 Activity of NRRL No. B-30145 againstselected plant pathogens in the slide germination assay. Alternariabrassicicola No Germination Alternaria dauci No Germination Botrytiscinerea No Germination Monilinia fructicola No Germination

Example 3

[0061] Activity of NRRL Against Plant Pathogens in Plant Bioassay Tests.

[0062] Activity of NRRL No. B-30145 was tested against tomato lateblight (Phytophthora infestans), tomato early blight (Alternariasolani), gray mold (Botrytis cinerea), turf brown patch (Rhizoctoniasp.), and peanut southern blight (Sclerotinia minor). All tests wereconducted under controlled environment in the laboratory with plantmaterial grown from seed under typical commercial greenhouse conditions.

[0063] Tomato Late Blight-Phytophthora infestans

[0064] The pathogen is grown on rye agar in standard petri dishes at 16°C. in the dark. Sporangia are collected by flooding the plate with waterand scraping the mycelium to dislodge the sporangia. The sporangialsuspension is passed through cheesecloth, quantified and adjusted to1.0×10⁴. Tomato seedlings at the 3^(rd) to 5^(th) leaf stage are sprayedto run-off with the fermentation sample of NRRL No. B-30145 using anartist airbrush at 40 psi. Treated seedlings are allowed to air dry atroom temperature for at least two hours then inoculated with thesporangial suspension by lightly spraying the upper surfaces of thetomato seedlings using a hand held sprayer. Inoculated seedlings areplaced in solid bottom flats filled with water and then are covered witha plastic dome to maintain leaf wetness. Flats are incubated at 20° C.with a 14-hr photoperiod for four days continuously covered by theplastic domes. Seedlings are then rated based on a disease rating scalefrom 0-5 with 0 equaling no symptoms, and 5 equaling 75% or more of thefoliage colonized by the pathogen. A typical example of a late blighttest is presented in Table 3. TABLE 3 Results of NRRL No. B-30145treated tomato seedlings against the late blight pathogen Phytophthorainfestans. Treatment Ave. D.I. Replications 1-4 Sample 990702 1.1 1.00.5 2.0 1.0 Sample 990709 1.1 1.0 2.0 1.0 0.5 Sample 990825 1.3 1.5 1.01.5 1.0 Sample 990913 1.0 1.0 1.0 1.5 0.5 Quadris 30 ppm 0.1 0 0.5 0 0Water Check 4.3 4.0 4.0 5.0 4.0

[0065] Samples are different fermentations of NRRL No. B-30145.

[0066] D.I. is Disease Index.

[0067] Tomato Early Blight-Alternaria solani

[0068] The pathogen is first grown on commercial Difco potato dextroseagar (PDA) at 22-25° C. under 14-hour lights until the entire plate iscovered. The fungus and the agar medium is then cut into small squaresapproximately 10 mm square and placed fungus side up on a specializedsporulation medium (S-Medium: 20 g sucrose, 20 g calcium carbonate, 20 gBacto-agar per liter). The S-Media plates are flooded with a thin layerof water and incubated 2-3 days at 22-25° C. under 14-hour lights untilfull sporulation of the pathogen occurs. Plates are then flooded withwater and the agar squares are scraped from the plate. The suspension ispassed through cheesecloth and the spores are quantified and adjusted to1.0×10⁵. Tomato seedlings at the 3^(rd) to 5^(th) leaf stage are thensprayed until run-off using an artist airbrush as described previously.Treated seedlings are allowed to dry and then inoculated with the sporesuspension. Seedlings are placed in flats and covered as describedpreviously and incubated at 25° C. with a 14-hour photoperiod. Seedlingsare rated based on a scale of 0-5 as previously described. Results froma typical test are presented in Table 4. TABLE 4 Activity of NRRL No.B-30145 against the early blight pathogen Alternaria solani. TreatmentsAve. D.I. Replications Test-1 Sample 990216 1.0 2.0 0.5 0.5 Quadris 60ppm 1.8 1.5 2.5 1.5 Water Check 4.0 3.0 4.0 5.0 Test-2 Sample 990216 1.11.5 1.0 1.0 1.0 Water Check 4.5 5.0 4.0 4.0 5.0

[0069] D.I. is Disease Index.

[0070] Pepper Gray Mold-Botrytis cinerea

[0071] The pathogen is grown on standard PDA under a 14-hour photoperiodat 22° C. until the fungal growth has completely covered the plate (7-9days). Spores are collected by flooding the plate with water and thengently scraping with a spatula to dislodge the spores. The sporesuspension is passed through cheesecloth and quantified and adjusted to1.5×10⁶. Pepper seedlings are grown until the 4^(th) to 6^(th) true leafstage and fermentation samples are sprayed on the upper leaf surfacesusing an artist airbrush as described previously. Treated seedlings areinoculated, placed in flats and covered with plastic domes. Flats areplaced at 20° C. under continuous darkness for 2.5 days. Seedlings arerated on a 0-5 scale as described previously. Table 5 depicts resultsfrom two typical tests. TABLE 5 Activity of NRRL No. B-30145 againstBotrytis cinerea Treatment Ave. D.I. Replications Test-1 Sample 9902161.4 1.5 1.5 1.5 1.0 Break 20 ppm 0.1 0 0 0.5 0 Water Check 4.0 4.0 4.03.0 5.0 Test-2 Sample 990216 1.9 1.5 2.0 2.0 2.0 Break 20 ppm 0.8 0 1.51.0 0.5 Water Check 4.5 4.0 5.0 5.0 4.0

[0072] D.I. is Disease Index.

[0073] Turf Brown Patch-Rhizoctonia sp.

[0074] Two ml of fermentation sample was added to each cell of a 6-cellpot of one-month old turf seedlings (Bentgrass). A 4 mm mycelial plug ofa 2-3 day old culture of Rhizoctonia sp. was placed under the soilsurface. Each treatment was replicated 6 times. Inoculted pots wereplaced in plastic flats and covered with a plastic dome. The flats wereplaced on a light rack (16Hr/day) and incubated at room temperature.Disease severity was evaluated after 5-6 days incubation and comparedwith the water treated control. The results indicated that NRRL No.B-30145 has a suppressive activity against Rhizoctonia (Table 6). TABLE6 The efficacy of NRRL No. B-30145 on turf disease caused by Rhizoctiniasp. Dilution Treatment Factor Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 301451x +* + + + + + 30145 ½x + + + + + + Water +++ +++ +++ +++ +++ +++

[0075] Peanut Southern Blight-Sclerotinia minor

[0076] Peanut seedlings at the first 2-leaf stage were treated with NRRLNo. B-30145 and a 4-mm mycelial plug is placed on the base of each stemafter the treated plants dried. Inoculated plants were incubated in adew chamber for 2 days before being placed in a plastic flat sealed witha cover dome. The flat was incubated on a light rack (16Hr/day) at roomtemperature for 10 days. Disease severity was assessed by comparing thetreated with the water control. The results (Table 7) indicated NRRL No.B-30145 whole broth at lx has some controlling activity againstSclerotinia minor. TABLE 7 The efficacy of NRRL No. B-30145 on peanutSclerotinia blight. Treatment Dilution Factor Rep 1 Rep 2 Rep 3 30145 1x+/− 0 +/− 30145 ½x ++ ++ + 30145 ¼x 0 ++ ++ Water +++ +++ +++

Example 5

[0077] Antifungal Metabolite Produced by NRRL No. B-30145.

[0078] The whole broth of NRRL No. B30145 was partitioned into ethylacetate, butanol and aqueous fractions. Each fraction was tested againstAlternaria brassicicola in a spore germination assay. The Alternariaspores were germinated in the presence of each sample in depressionmicroscope slides containing 40 μl of sample and 20 μl of pathogenspores. Approximately 16 hours later the spores are observed under amicroscope to see if they have germinated. No germination (score of 0)compared to the water control (100% germination and growth=score of 5)indicates activity of the sample being tested. Results of the Alternariagermination assay with different NRRL No. B-30145 fractions are shownbelow (score on a 0 to 5 rating as above). Score Fraction Rep 1 Rep 2Rep 3 Ethyl acetate 3 nd 4 n-butanol 0 0.2 1 Aqueous 0 5 5 Whole broth 00.2 0 Water Check 5 5 5

[0079] The metabolite is clearly in the butanol soluble fraction and isnot readily extractable in ethyl acetate. Other characteristics of themetabolite were determined. The molecule was shown to pass through a10,000 molecular weight cut off filter indicating the metabolite issmaller than 10,000 daltons. The activity was not lost when treated withbase or upon heating to 80 degrees C. for one hour. The activity waslost when treated with acid (the score against Alternaria increased from0 to 5).

[0080] Fractionation of the butanol extract on octadecylsilane bonded tosilica gel (ODS) flash chromatography using an acetonitrile (ACN)/watergradient with 0.01% trifluoroacetic acid (TFA) yielded an activefraction eluting with 50% acetonitrile/water with 0.01% TFA. Fractionswere tested in an Alternaria germination assay for activity (0-5 ratingscale). Fraction Score ODS 10% ACN 4 ODS 20% ACN 5 ODS 50% ACN 0.5 ODS100% ACN 5 Water Check 5

[0081] Further purification by ODS HPLC yielded 2 active components(Fraction 6 and 7) from an isocratic elution with 31% acetonitrile inwater with 0.02% TFA). Fraction Score HPLC Fr. 1 5 HPLC Fr. 2 5 HPLC Fr.3-5 4 HPLC Fr. 6 3 HPLC Fr. 7 2 HPLC Fr. 8 5 HPLC Fr. 9 5 Water Check 5

[0082] The HPLC chromatogram of the active 50% acetonitrile/water with0.01% TFA flash chromatography fraction and the HPLC chromatograms ofthe active fractions 6 and 7, including UV spectra of the activeprinciples, are shown in FIGS. 1 and 2.

[0083] NRRL No. B-30145 most closely matched Streptomyces mashuensis by16S RNA sequencing. Unlike the antibacterial metabolites typicallyassociated with S. mashuensis, the fungicidal activity of NRRL No.B-30145 was extractable with butanol. S. mashuensis is known to producestreptomycin, which is a water-soluble antibacterial compound. Anotherantibiotic produced by S. mashuensis, monazomycin (Akasaki et al. 1963),does not display a shoulder at 215-220 nm as does the fungicidal activefractions of NRRL No. B-30145.

[0084] Antifungal compounds have also been found in the closely relatedand possibly synonymous species Streptomyces griseocarneum (AmericanType Culture Collection). These include porfiromycin (Claridge et al.,1986), a purple compound whose corresponding UV spectrum is not seen inthe active fraction of NRRL No. B-30145 and the Heptaenes trichomycin(Komori and Morimoto, 1989) and griseocarnin (Campos et al., 1974),whose corresponding UV spectra are also not present in the activefraction. The fungicidal active is also not neutramycin, which isextractable with ethyl acetate (Mitscher and Kunstmann, 1969).

Example 6

[0085] Additional Methods for Further Purification of the AntifungalMetabolite of NRRL No. B-30145

[0086] Method A

[0087] The freeze-dried whole broth culture was re-suspended in water(2.0 L) and loaded onto a column containing a non-ionic polymeric resin(Supelco Sepabead SP-207; 26×3.0 cm) equilibrated in water. The columnwas washed with water (200 mL) and then with a gradient of aqueousmethanol as follows: (1) 20:80 methanol/water (200 mL), (2) 40:60methanol/water (200 mL), (3) 60:40 methanol/water (200 mL), 80/20methanol/water (200 mL), and (5) methanol (200 mL).

[0088] Bioassay results (germination assay with Monilinia fructicolaand/or Alternaria brassicicola) indicated that all fractions wereactive. Each fraction was individually fractionated onoctadecylsilane-bonded silica gel (ODS) HPLC using anacetonitrile/methanol/water (TOSOHASS ODS-80TS; 10 μm, 21.5×30 cm.Solvent system: solvent A: acetonitrile/methanol/water 25:5:65, solventB: acetonitrile/methanol/water 65:5:30. Gradient: start at 0 min withsolvent A and hold for 25 min. Then increase solvent B to 35% over 50min. Flow=6.0 mL/min). All fractions yielded approximately the same HPLCprofile with the activity located at two regions: peak A (t˜55-63 min)and peak B (t˜65-70 min). Peak B was further fractionated on anotherreversed-phase HPLC column (Phenomenex Luna Phenyl-Hexyl; 5 μm, 250×10mm. Solvent system: solvent A: acetonitrile/methanol/water 25:5:65,solvent B: acetonitrile/methanol/water 65:5:30. Gradient: start at 0 minwith solvent A and hold for 15 min. Then increase solvent B to 25% over25 min. Flow=2.0 mL/min). One major component was isolated; however,analytical HPLC analysis indicated a high-UV absorbing contaminant thatco-eluted with active metabolite. Therefore, an alternative purificationmethod was employed (method B).

[0089] Method B

[0090] Alternatively, the homogenized cell-free whole broth culture ispassed through non-ionic polymeric resin (Supelco Diaion HP-20), washedwith water, and then methanol. The methanol eluate is further separatedby reversed-phase HPLC (HP Zorbax Eclipse XDB-C8; 5 μm, 150×4.6 mm.Solvent system: solvent A: acetonitrile/methanol/water 25:5:65, solventB: acetonitrile/methanol/water 65:5:30. Gradient: start at 0 min withsolvent A and increase solvent B to 3% in 20 min. Flow=0.8 mL/min) toafford the same active peaks observed in method A (peaks A and B) andconfirmed by analytical HPLC using UV and MS detection. An HPLC trace isshown in FIG. 3.

Characteristics of Active Metabolites of NRRL No. B-30145

[0091] The impure fraction obtained from method A provided some initialinformation about the nature of the active metabolite. LC MS indicated amolecular weight [M+H⁺]=892.6 and the UV spectrum displays a shoulder at215-220 nm. 1D and 2D NMR suggests at least 2 propargyl alcohol segments[C═C—CH(OH)], several oxygenated methine carbons (X—CH—Y), and apossible sugar moiety. ¹H and ¹³C NMR are shown in FIGS. 4 and 5respectively.

[0092] Even though method B has not provided sufficient quantities forNMR analysis, this method yielded cleaner peaks in sufficient amountsfor analysis by HPLC (octyl bonded silica gel) using UV and MS detectionmethods. Two major peaks (peak A and B) were obtained that matched thesame compounds identified as the active metabolites using method A (seeFIG. 3). The UV spectra of all compounds presented a shoulder at 215-220nm. LC MS of peak A indicated the presence of at least three (3)compounds with the following molecular weights [M+H^(+]=)866.5, 882.5,and 898.4 (see FIG. 6). Similarly, peak B showed at least three (3)compounds with molecular weights [M+H⁺]=892.5, 908.5, and 924.5 (seeFIG. 7).

[0093] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

REFERENCES

[0094] Akasaki et al., “Monazomycin, a new antibiotic produced by astreptomyces,” J. Antibiotics, vol. 16, pp 127-131 (1963).

[0095] Axelrood et al., “Douglas-fir root-associated microorganism withinhibitory activity towards fungal plant pathogens and human bacterialpathogens,” Can. J. Microbiol., vol. 42, pp. 690-700 (1996).

[0096] Campos et al., “[Griseocarnin, an antifungal antibiotic isolatedfrom Streptoverticillium griseocarneum (IA-7527)],” Rev Inst Antibiot(Recife), vol. 14(1-2), pp. 91-100 (1974) (English Summary only).

[0097] Chamberlain and Crawford, “In vitro and in vivo antagonism ofpathogenic turfgrass fungi by Streptomyces hygroscopicus strains YCED9and WYE53,” J of Industrial Microbiol Biotechnol, vol. 23, pp. 641-646(1999).

[0098] Claridge et al., “New mitomycin analogs produced by directedbiosynthesis,” J. Antibiotics vol. 39, pp. 437-446.

[0099] Crawford, “Use of Streptomyces bacteria to control plantpathogens,” U.S. Pat. No. 5,527,526.

[0100] Komori and Morimoto, “Isolation of the aromatic heptaenicantibiotics trichomycin A-F by high-performance liquid chromatography,”J. Chromatogr, vol. 481, pp 416 (1989).

[0101] Lechevalier, H. A., and S. A. Waksman, “The actinomycetes.IIIAntibiotics of actinomycetes”, The Williams & Wilkins Co., Baltimore,p430ff, (1962).

[0102] Lechevalier, M. P, “Actinomycetes in agriculture and forestry,”In M. Goodfellow, S. T. Williams, and M. Mordarski (ed.), Actinomycetesin biotechnology. Academic Press, Inc., New York, p.327-358 (1988).

[0103] Mitscher, L. A. and M. P. Kunstmann, “The structure ofneutramycin,” Experientia vol. 25, pp. 12-3 (1969).

[0104] Reddi, G. S., and A. S. Rao, “Antagonism of soil actinomycetes tosome soil-borne plant pathogenic fungi,” Indian Phytopathol. Vol. 24,pp. 649-657 (1971).

[0105] Rothrock, C. S., and D. Gottlieb, “Role of antibiosis inantagonism of Streptomyces hygroscopicus var. geldanus to Rhizoctoniasolani in soil,” Can. J. Microbiol., vol. 30, pp. 1440-1447, (1984).

[0106] Schwinn et al., “Advances In Plant Pathology: Phytophthorainfestans, The Cause Of Late Blight Of Potato,” (Academic Press, SanDiego, p. 24⁴ (1991).

[0107] Suh, “Antifungal biocontrol agents, a process for preparing andtreating the same,” International Patent Publication Number WO 98/35017.

[0108] Yuan and Crawford, “Characterization of Streptoimyces lydicusWYEC108 as a potential biocontrol agent against fungal root and seedrots,” Appl. Env. Microbiol. vol. 61, pp. 3119-28 (1995).

What is claimed is:
 1. A metabolite produced by Streptomyces sp. strainNRRL No. B-30145 and mutants thereof having all the identifyingcharacteristics of NRRL No. B-30145 and that exhibits activity againstplant pathogenic fungi.
 2. The metabolite of claim 1, wherein themetabolite has a molecular weight [M+H⁺] between about 925 to about 865.3. The metabolite of claim 2, wherein the molecular weight is selectedfrom the group consisting of 866.5, 882.5, 898.4, 892.5, 908.5 and924.5.
 4. The metabolite of claim 1, wherein the metabolite is heat andbase stable, is acid labile and has a molecular weight [M+H⁺] betweenabout 925 to about
 865. 5. The metabolite of claim 4, wherein themolecular weight is selected from the group consisting of 866.5, 882.5,898.4, 892.5, 908.5 and 924.5.
 6. The metabolite of claim 1, wherein themetabolite has a chromatogram at 220 nm shown in FIG.
 3. 7. Themetabolite of claim 1, wherein the metabolite exhibits UV absorptionbetween about 215 nm and 220 nm.
 8. The metabolite of claim 1, whereinthe metabolite has a ¹H Nuclear Magnetic Resonance spectra shown in FIG.4.
 9. The metabolite of claim 1, wherein the metabolite has a ¹³CNuclear Magnetic Resonance spectra shown in FIG.
 5. 10. The metaboliteof claim 1, wherein the metabolite comprises one or more moleculesselected from the group consisting of propargyl alcohol segments[C═C—CH(OH)], oxygenated methine carbons (X—CH—Y) or a sugar moiety. 11.The metabolite of claim 10, wherein the metabolite comprises at leasttwo propargyl alcohol segments [C═C—CH(OH)].
 12. A compositioncomprising the metabolite of claim 1 and a carrier.
 13. A compositioncomprising more than one metabolite of claim 1 and a carrier.
 14. Thecomposition of claim 12, further comprising at least one chemical orbiological pesticide.
 15. The composition of claim 13, furthercomprising at least one chemical or biological pesticide.
 16. Thecomposition of claim 12, wherein the composition is formulated from thegroup consisting of a wettable powder, a granule, an aqueous suspension,and emulsifiable concentrate and a microencapsulated formulation. 17.The composition of claim 13, wherein the composition is formulated fromthe group consisting of a wettable powder, a granule, an aqueoussuspension, and emulsifiable concentrate and a microencapsulatedformulation.
 18. A method for protecting or treating plants, fruit, androots from fungal infections comprising applying an effective amount ofthe metabolite of claim 1 to the plant, fruit or root.
 19. The method ofclaim 18, wherein the infections are caused by a fungus selected fromthe group consisting of Alternaria solani, Botrytis cinerea, Rhizoctoniasp., Sclerotinia sp., and Phytophthora sp.
 20. The method of claim 18,wherein more than one metabolite of Streptomyces sp. NRRL No. B-30145strain that exhibits activity against plant pathogenic fungi is applied.21. The method of claim 18, wherein the metabolite has a molecularweight [M+H⁺] between about 925 to about
 865. 22. The method of claim21, the molecular weight of the metabolite is selected from the groupconsisting of 866.5, 882.5, 898.4, 892.5, 908.5 and 924.5.
 23. Themethod of claim 18, wherein the metabolite is heat and base stable, isacid labile and has a molecular weight [M+H⁺] between about 925 to about865.
 24. The method of claim 23, wherein the molecular weight isselected from the group consisting of 866.5, 882.5, 898.4, 892.5, 908.5and 924.5.
 25. The method of claim 18, wherein the metabolite has achromatogram at 220 nm shown in FIG.
 3. 26. The method of claim 18,wherein the metabolite exhibits UV absorption between about 215 nm and220 nm.
 27. The method of claim 18, wherein the metabolite has a ¹HNuclear Magnetic Resonance spectra shown in FIG.
 4. 28. The metaboliteof claim 18, wherein the metabolite has a ¹³C Nuclear Magnetic Resonancespectra shown in FIG.
 5. 29. The method of claim 18, wherein themetabolite is applied as a formulation selected from the groupconsisting of wettable powders, granules, aqueous suspensions,emulsifiable concentrates or microencapsulations.
 30. The method ofclaim 29, further comprising applying an effective amount of at leastone chemical or biological pesticide.
 31. The method of claim 29,wherein the formulation comprises more than metabolite.
 32. Anantifungal composition comprising a metabolite produced by Streptomycesand isolated according to a method comprising: (a) loading a whole brothculture of Streptomyces sp. strain NRRL No. B-30145 or mutants thereofhaving all the identifying characteristics of NRRL No. B-30145 onto anon-ionic absorbent polymeric resin; (b) eluting the metabolite with analcohol; (c) screening the eluent of step (b) with a bioassay forfractions of the eluent exhibiting antifungal activity; (d) loading thefractions of the eluent exhibiting antifungal activity of step (c) on aHPLC column; and (e) eluting the metabolite with an organic solvent. 33.The composition of claim 32, wherein the eluent of step (b) is methanolor a gradient of aqueous methanol.
 34. The composition of claim 32,wherein the bioassy of step (c) is selected from the group consisting ofthe agar diffusion assay or slide germination assay.
 35. The compositionof claim 32, wherein the organic solvent of step(e) is anacetonitrile-water gradient.
 36. A method for protecting or treatingplants, fruit, and roots from fungal infections comprising applying aneffective amount of the composition of claim 32 to the plant, fruit orroot.
 37. The method of claim 32, wherein the infections are caused by afungus selected from the group consisting of Alternaria solani, Botrytiscinerea, Rhizoctonia sp., Sclerotinia sp., and Phytophthora sp.
 38. Themethod of claim 32, wherein the Streptomyces sp. strain NRRL No. B-30145is applied as a formulation selected from the group consisting ofwettable powders, granules, aqueous suspensions, emulsifiableconcentrates or microencapsulations.
 39. The method of claim 32, furthercomprising applying an effective amount of at least one chemical orbiological pesticide.